Antibody therapeutic agents show high efficacy and less side effects together in the treatment of various diseases compared to chemical agents, and thus have become the research objectives of global pharmaceutical companies and bioengineering companies. At present, a large number of antibody therapeutic agents are used in clinical applications, and many therapeutic agent candidates are under clinical trials.
However, despite such advantages, the antibody therapeutic agents have problems, including low tissue penetrability due to their large molecular weight, high product costs due to complicated production processes, and entry barriers created by existing patents. Due to such problems, artificial antibodies to replace the antibody therapeutic agents have recently been developed. Many studies have revealed that such artificial antibody scaffold proteins have an advantage over the antibody therapeutic agents in that they penetrate cancer tissue with greatly increased efficiency, suggesting that therapeutic effects of the artificial antibodies can be improved.
Under this background, the present inventors successfully developed a repebody which is a non-antibody protein scaffold capable of replacing existing antibodies. It was shown that the repebody had a size equal to about ⅕ of antibody, was produced in large amounts in E. coli, and showed little or no immunogenicity in animal studies. In addition, it was demonstrated that the repebody has very high thermal and pH stabilities, and its ability to bind to a target can be very easily increased to picomole levels, and its specificity for a target is very high (Korean Patent Application No. KR2013-0098089A).
Epidermal growth factor receptor (EGFR) is a disease inducer verified to be associated with colorectal cancer and a variety of cancers, and 90% or more of total colorectal cancer cases are known to be malignant tumors of epidermal origin. Overexpression and mutation of the EGFR are found in 50-90% or more of such cancers, and abnormalities in signal transduction systems by the EGFR are found commonly in the majority of cancers, and for this reason, the EGFR has been recognized as a major target in the development of anticancer agents. Furthermore, up to date, signal transduction systems through the EGFR are most clearly known compared to other signal transduction systems, and thus studies on the development of therapeutic agents that target the EGFR have been most actively conducted.
At present, drugs that target the extracellular domain of the EGFR include Cetuximab that is a chimeric monoclonal antibody drug. However, Cetuximab was reported to have low binding affinity and therapeutic effects compared to other monoclonal antibody drugs. In addition, it was reported to Cetuximab, when administered continuously, shows drug resistance, and causes side effects due to drug toxicity and side effects due to the chimeric antibody. To overcome such problems, it has been attempted to develop a new therapeutic method (such as multi-drug therapy) and new drugs (such as Nimotuzumab). Therefore, there is a need to develop EGFR-targeting therapeutic agents having a new concept, which overcome the limitations of existing drugs.
The present inventors successfully prepared a specific protein binder for various disease-related target proteins by use of the repebody scaffold, and verified through a cell-based method that the specific protein binder has biological inhibitory effects. However, studies on the application of the specific protein binder are still in the opening stage, and thus additional studies are required. In addition, the identification of lead substances, which bind to various receptors overexpressed in cancer cells, and the application thereof, have not yet been reported.
Under this background, the present inventors have screened a novel protein having a specific binding affinity for epidermal growth factor receptor (EGFR), based on a random mutation library constructed through analysis of the modularity (structural feature) and the overall structure of the repebody, in order to identify a protein that binds specifically to the extracellular domain of the EGFR, which is overexpressed in various cancers, by use of the repebody scaffold, and have found that the binding affinity of the protein can be increased by a method of increasing the affinity based on repeat modules, thereby completing the present invention.